chapter 7: in vitro and in vivo supramolecular biomembrane

Chapter 7

In vitro and In vivo supramolecular biomembrane engineering using a lipidated coiled-coil motif

A simple and generic tool for the efficient formation of coiled coils at the surfaces of

cells and zebrafish embryos is presented here. A pair of complementary coiled-coil-

forming peptides, denoted E and K, were synthesized and conjugated to a cholesterol

anchor. The resulting lipidated peptides entered into CHO cell membranes

spontaneously in vitro and into the skin of zebrafish embryos in vivo. Addition of the

acetylated complementary peptide resulted in efficient coiled coil formation at the

surface of cells and zebrafish embryos. As the coiled coil unit acts as a molecular

velcro, this supramolecular tool enables the modification of biomembranes with a

wide range of molecular constructs. As a case study, the docking of liposomes to cell

membranes and zebrafish skin was investigated. Liposomes bearing the

complementary peptide efficiently docked at these biological interfaces through coiled

coil formation. Importantly, this is the first time that in vivo membrane modification

has been achieved. It is envisaged that this tool will aid the study of membrane

processes by modulating the chemistry of these bio-interfaces. Furthermore,

applications in the area of pharmacology and drug delivery such as high-throughput

screening of drugs are expected.

This work is published: H. Zope, F. Versluis, A. Ordas, J. Voskuhl, H. P. Spaink and

A. Kros, Angew. Chem. Int. Ed. Manuscript accepted for publication.

Chapter 7

Introduction

The molecular building blocks available in biological systems are able to self-

assemble into well-defined structures in an extremely controlled manner. These

structures need to be flexible and adaptive to the environment to enable them to carry

out their function in a regulated manner. Therefore, nature uses multiple weak

interactions (e.g. hydrogen bonding and van der Waals interactions) to act as the glue

to hold these structures together. When many weak interactions cooperatively

combine, relatively stable entities are produced, which retain the ability to ‘respond’

to external stimuli such as fluctuations in ion concentration, pH and temperature. For

many years, nature has been a source of inspiration for supramolecular chemistry.1-4

Here, scientists design relatively simple molecules which assemble into functional

materials with well-defined properties, typically following the bottom up approach.5-8

Recent progress has resulted in molecular systems which are responsive to multiple

stimuli and are therefore highly controlled, emulating nature ever more closely. A

relatively new branch of development is the application of supramolecular constructs

in an in vitro and in vivo environment, to directly study and influence biological

processes in live cells.

Introduction of supramolecular systems into a biological environment allows for the

placement of chemically tailored systems into, for example, cell membranes. This

enables the modification or regulation of cellular behavior through artificial signals.9

From a supramolecular chemistry point of view, two types of approach can be pursued

to introduce chemical species into a cell membrane: 1) specific binding of guest

molecules to membrane anchored biomolecules such as native proteins10 or 2) non-

specific labeling of membranes with the aid of hydrophobic and electrostatic

interactions or through a chemical cross linker.11-13 Lipidated peptides are particularly

good candidates for application in biological systems as their aggregation behavior

can be controlled by carefully balancing the hydrophobicity of the anchor and the

hydrophilicity of the cargo,14 which aids the incorporation of lipidated peptides into

membranes. By employing a coiled coil motif as the peptide segment, a highly

specific recognition system can be introduced into live cells. The coiled coil motif

acts as “molecular Velcro” and can thus be used to link distinct molecular constructs

together.15-20 An example of specific labeling of proteins through coiled coil formation

was recently supplied by Matsuzaki et al.21 Surface modification through non-specific

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In vitro and in vivo biomembrane engineering using a lipidated coiled-coil motif

binding of polymers to cell membranes has also been studied, for example by Ijiro et

al.22 Lipid grafted polymers adhered to cell membranes and could potentially act as a

scaffold to which a wide range of functional moieties could be attached, thereby

intervening in the chemistry of the cell. Furthermore, cationic graft copolymers have

also been shown to interact electrostatically with cell membranes, resulting in

chemically altered cell membranes.23 Whereas all these examples illustrate that in

vitro membrane functionalization is a highly rewarding strategy, examples of efficient

in vivo strategies are currently still lacking. Therefore, it is our goal to transiently

modify lipid membranes through specific supramolecular interactions in an in vitro

and in vivo environment. This will enable studies of membrane related processes by

modulating the chemistry at these interfaces. For this purpose, a pair of

complementary coiled coil forming lipidated peptides E and K,15, 24, 25 was used to

specifically introduce a non-covalent and bio-orthogonal recognition motif to

influence the chemistry of biological membranes (Scheme 1).

Scheme 1. Schematic representations of A) the lipidated E and K peptides, a CPE

decorated liposome and the fluorescently labelled E peptide, B) anchoring of CPK

into CHO cell membranes and subsequent coiled coil formation with fluorescently

labeled E or liposomes decorated with E peptides and C) anchoring of CPK into the

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skin of a zebrafish embryo and subsequent coiled coil formation with fluorescently

labeled E or liposomes decorated with peptide E.

Here, a generic supramolecular tool which allows us to rapidly and efficiently form

coiled coil motifs at the surface of biological membranes was developed. This is of

interest as a wide range of molecular constructs can be introduced to the surface of the

cell through a specific coiled coil formation. Coiled coil forming peptides E

[(EIAALEK)3] and K [(KIAALKE)3] 26-29 were first covalently conjugated to a

PEG12 spacer. Subsequently, a cholesterol moiety was coupled to the pegylated

peptides yielding CPE and CPK (Scheme 1A).30 The cholesterol moiety allows for the

immediate insertion of the lipidated peptides into membranes through hydrophobic

interactions and the PEG12 moiety was incorporated to aid efficient molecular

recognition between the peptide segments E and K. Recently, it was shown that upon

the addition of micellar solutions of either CPE or CPK to plain liposomes, the

lipidated peptides spontaneously inserted into liposomal membranes.30 In the present

study, CHO cell membranes and the skin of zebrafish embryos were modified with

coiled coil forming peptides by the addition of a micellar solution of CPE or CPK

resulting in immediate incorporation of these amphiphiles into the membranes.

Subsequently, the complementary peptide was added, resulting in coiled coil

formation. This system thus enables the docking of a wide variety of molecular

constructs through coiled coil formation (Scheme 1B and C).

Results and Discussion

In order to establish coiled coil formation at the surfaces of Chinese hamster ovarian

(CHO) cells, it was first confirmed that the cholesterol anchor entered into the

membranes of the cells. After the addition of a 5 µM solution of fluorescein labeled

CPE and incubation for a few minutes, confocal microscopy was used to image the

treated cells. As expected, the cells showed highly fluorescent membranes, indicating

that CPE was efficiently incorporated into the cell membrane (Figure 1). Surprisingly,

some internalization of CPE was observed. A similar effect was observed by Irvine et

al., who used a diacyllipid (C18) and cholesterol to anchor the oligonucleotides into

cell membranes.31 These lipidated oligonucleotides are able to anchor on the cell

membrane in 30 min whereas lipidated peptides could anchor in less than 5 min. One

explanation for this is that as oligonucleotides are more anionic as compared to the

162


peptides used in this study, electrostatic repulsion might delay incorporation into the

membrane. It was also found that incorporating a PEG24 spacer between the

oligonucleotide and lipid segments drastically reduced the insertion capability; a

PEG12 spacer did not hinder membrane insertion. Future work in our group will focus

on these issues in order to have a better understanding of peptide amphiphile

membrane insertion.

Figure 1. Confocal images of CHO cells. The cells were treated with the nuclear stain

Hoechst for 30 min followed by incubation with fluorescently labeled CPE at 37 °C.

Images were obtained A) 0 min and B) 10 min after the addition of fluorescent CPE (5

µM). The inset shows a magnified image, revealing some internalization of the

lipopeptide. The scale bar represents 25 µm.

Subsequently, it was investigated whether the peptides at the CHO cell surface were

accessible and capable of forming coiled coils with the complementary peptide. For

this, cells were treated with a CPK solution of a final concentration of 5 μM, which is

the standard concentration for the lipidated peptides used throughout this study. A 5

minute incubation time was followed by washing with HBSS buffer to remove any

unbound CPK. Next, a solution containing 2.5 µM carboxyfluorescein labeled peptide

E was added to the cells and incubated for 10 minutes. Three more washes with HBSS

were performed to again remove any non-bound peptide E. Confocal imaging of the

CHO cells revealed that the fluorescent signal was located at the cell membranes

(Figure 2). In a control experiment the addition of CPK was omitted and as a result

the cells showed no fluorescence (Appendix, Figure S1). These observations strongly

suggest that the coiled coil formation between the membrane bound peptide E and

peptide K is efficient. Additionally, the opposite route could also be taken, i.e.,

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addition of CPE first, followed by treatment with fluorescent K peptides, which also

yielded specific coiled coil formation, as observed by the fluorescence located at the

membranes (Appendix, Figure S2). These results show that both CPK and CPE can

be used to functionalize the CHO cell membrane. In contrast, the protein engineering

approach only led to coiled coil formation when the engineered membrane protein

contained peptide E, but not the other way around. 32 Furthermore, our system

potentially allows for the modification of cell membranes of various cell lines without

the aid of laborious protein engineering protocols. Additionally, our supramolecular

method is fast and the necessary peptides can be synthesized within one day, as

opposed to the protein engineering approach, where the incorporation of the modified

receptor could take significantly more time.

Figure 2. (A, B, C) Confocal microscopy images (A: bright field, B: fluorescence and

C: overlay) indicating coiled coil formation between E and K at the surface of CHO

cells. First, a 5 µM solution of CPK in HBSS was added to the cells, 5 min incubation

time. Subsequently, fluorescently labeled (carboxyfluorescein) E peptides (2.5 µM)

were added and incubated for 5 minutes. Imaging took place after three washes with

HBSS. Experiments were performed at 37 °C. The scale bar represents 50 µm. (D, E,

F) Confocal microscopy images (D: bright field, E: fluorescence and F: overlay)

indicating docking of liposomes at the surface of CHO cells. First, a 5 µM solution of

CPK in HBSS was added to the cells, 5 min incubation time. Subsequently, CPE

decorated liposomes (0.25 mM total lipid concentration and 1 mol% CPE) with 1

mol% DHPE-TR were added to the cells. All the experiments were performed at 37

°C. The scale bar represents 25 µm.

164


These initial studies demonstrated that coiled coils can be formed at the surface of

CHO cells by simply incubating a cholesterol peptide with the CHO cells and

subsequently adding the complementary peptide. This system enables the docking of

various molecules and nanoparticles to the cells. It was recently demonstrated that 100

nm liposomes can be immobilized at interfaces mediated by coiled coil formation

between peptides E and K.33 Therefore it was of interest to study whether liposome

docking is also possible at a biological interface. Instead of adding a fluorescent

peptide to the pre-treated cells, the complementary peptide was incorporated into

liposomes (Figure 2). After membrane functionalization with CPK, fluorescent

liposomes, containing 1 mol% DOPE-LR and 1 mol% CPE were incubated with the

cells for 5-10 minutes. After 3 washes, confocal images of the cells were obtained

(Figure 2). The images show increased fluorescence at the cell membranes.

Furthermore, with the CPE liposomes by themselves, i.e. without the presence of

peptide K at the cell membranes, no docking to the cells was observed (Appendix,

Figure S3). This shows that large supramolecular assemblies can be docked onto

membranes in an in vitro environment, using the orthogonal non-covalent coiled coil

motif. These findings enable future applications such as biophysical studies or

pharmaceutical research, for example adjuvant modified cells can be injected inside

animals, which might lead to cell therapy applications.34

Preclinical studies are a required step in translating innovations from a laboratory

setting to the clinic. Recent restrictions on animal testing have increased the demand

for alternative animal models. Therefore, zebrafish have become an attractive model

organism in recent years as they can be utilized as a tool for medical research bridging

the gap between cell-based assays and mammalian models.35 Due to its low-cost of

husbandry, high reproductive capability, optical transparency of the embryos, and the

ease of experimental manipulation, zebrafish might be a valuable tool in the

development of novel drug delivery systems for human disease therapy.36

Consequently, it was examined whether our coiled coil based system could also

function in an in vivo zebrafish embryo environment. To the best of our knowledge,

surface modification of zebrafish has not been studied before and therefore it was

investigated whether our surface engineering system is functional in this in vivo

environment (Scheme 1C). As the epidermis of zebrafish embryo consists only of

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living cells, unlike terrestrial vertebrates in which dead, keratinized cells are present,

it is possible to functionalize the whole organism.

One day-old zebrafish embryos were pre-treated with a solution of CPK in egg water.

Next, fluorescently labeled peptide E was added and incubated with the zebrafish

embryos for 30 minutes, followed by 3 washes with egg water prior to imaging.

Fluorescent stereomicroscope imaging revealed that the skin of the embryos was

highly fluorescent (Figure 3A-C). This is a strong indication that efficient coiled coil

formation was accomplished in vivo. Furthermore, the control experiment in which

the embryos were not pre-treated with CPK showed no significant fluorescence

(Appendix, Figure S4). This strongly indicates that CPK efficiently decorated the

skin of the zebrafish and that the peptide was available for coiled coil formation with

its complementary peptide. Similar to in vitro experiments, functionalizing zebrafish

embryos with CPE followed by incubation with fluorescently labeled K peptide was

also attempted. Surprisingly, this alternative route did not yield efficient coiled coil

formation on the skin of the zebrafish embryos, which is in contrast to the cell-based

study. Fish are known to have a different lipid composition than terrestrial vertebrates,

as they contain high levels of polyunsaturated fatty acids.37, 38 Presumably, this lipid

composition favors the positively charged K peptide over the negatively charged E

peptide. However, further studies are necessary to elucidate the difference in the

behavior of the peptides at the surface of CHO cells versus zebrafish embryos.

Figure 3. (A, B, C) Coiled coil formation between CPK and fluorescently labelled E

peptides on the skin of zebrafish embryos, as indicated by fluorescent

stereomicroscope images of 1 day-old zebrafish embryos. The embryos were pre-

treated with 5µM CPK in egg water, followed by incubation with 2.5 µM

complementary fluorescent peptide E for 30 minutes at 34°C (A: bright field, B:

166


fluorescence, C: overlay). (D, E, F) Docking of liposomes on the skin of zebrafish

embryos through coiled coil formation. Fluorescent stereomicroscope images of 2

day-old zebrafish embryos pre-treated with 5µM CPK in egg water followed by

incubation with 0.25 mM liposomes bearing 1 mol% CPE and 1 mol% DHPE-TR on

the membrane for 30 minutes at 34°C (A: bright field, B: fluorescence, C: overlay).

Embryos are shown in lateral view, anterior to the left.

As a final investigation, docking of liposomes, as previously demonstrated for the

CHO cells, could also be achieved in vivo, on the skin of 2 day-old zebrafish embryos

was studied. In each group, embryos were selected and these embryos were first

incubated with CPK in egg water for 30 min at 34 °C. After 30 min. embryos were

washed and then incubated with 1 mol % CPK decorated fluorescently labeled

liposomes for 30 min at 34 °C. Upon the addition of fluorescent E decorated

liposomes to zebrafish that were pre-treated with CPK, the skin lit up brightly,

revealing the docking of liposomes to the skin of the embryos (Figure 3D-F). Control

experiments in which no CPK was administered displayed no significant fluorescence,

showing that liposome docking can be attributed to the specific coiled coil

interactions between peptides E and K (Appendix, Figure S5). As zebrafish have

been intensively studied as a model for various infectious diseases,39, 40 this study

enables future studies in which infected zebrafish embryos are treated using

therapeutic liposomes controlled by supramolecular coiled coil interactions.

Liposomes contain both hydrophilic and hydrophobic regions which enables a wide

range of drug encapsulation possibilities, which could be controllably released into

the zebrafish embryos. Therefore, in future studies a variety of therapeutic substances

will be screened, to investigate their effect on a range of diseases.41

Conclusions

In conclusion, a generic tool to modify the surface of live cells and zebrafish embryos

in a fast and efficient manner was designed. By using a supramolecular approach,

employing an orthogonal coiled coil forming peptide pair, specific interactions

between peptides E and K lead to coiled coil formation at the membrane of CHO cells

and the skin of zebrafish embryos. This method can be used to dock a wide range of

materials at a high local concentration to CHO cells and zebrafish embryos, which

was demonstrated by liposome docking in vitro and in vivo. This generic method

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holds much promise as an elegant and fast tool in supramolecular biomembrane

engineering and drug delivery. It also enables biophysical studies of biological

processes occurring at membranes. In future work, the aim is to introduce specificity

into the system. This might be achieved by constructing mixed micelles containing

our lipidated peptides and lipidated aptamers. The aptamers can be designed to attach

to specific cell types. In this way, it might be viable to deliver the lipidated peptides to

a specific cell type. Subsequent addition of liposomes that are decorated with the

complementary peptide should give cell specific liposome docking.

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Experimental section

Materials

The Fmoc-protected amino acids were purchased from Novabiochem. The Sieber

Amide resin was purchased from Agilent Technologies. Fmoc-NH-PEG12-COOH was

purchased from IRIS Biotech. DOPE, cholesterol and DOPC were obtained from

Avanti Polar Lipids. Cholesteryl hemisuccinate and 5,6-Carboxyfluorescein were

obtained from Sigma-Aldrich. 8 well plate Lab-tek was purchased from Thermo

Scientific, USA. DMEM media was obtained from PAA-the cell culture company. 3-

aminobenzoic acid (Tricaine) was purchased from Sigma-Aldrich.

General methods

Peptide synthesis

The cholesterol modified peptides were synthesized as described elsewhere in

chapter 4. Synthesis of fluorescently labeled peptides was performed on rink amide

resin. After initial Fmoc deprotection using 20% peperidine in DMF, Fmoc-Lys(boc)-

COOH was coupled, using 4 eq. of DIC and 4 eq. of HOBT, for 1 hour. Next, the side

chain Boc group was removed using TFA/ DCM (50:50 v/v) for 30 mins. The resin

was then neutralized using DIPEA/NMP (10:90 v/v) for 10 mins (3x). 5(6)-

Carboxyfluorescein (Sigma Adrich) was coupled using 4 eq. of DIC and 4 eq. of

HOBT dissolved in DMF and coupling was performed overnight. The resulting

fluorescently labeled resin was used to synthesize fluorescently labeled acetylated

peptide E and K along with fluorescently labeled CPE using standard Fmoc chemistry

procedure described earlier.30

Peptide and liposome sample preparation

Lipid (DOPC/DOPE/CH, molar ratios 2:1:1, total concentration 1 mM in CHCl3) and

peptide (50 µM in 1:1 CHCl3/MeOH) stock solutions were prepared. Peptide samples

were prepared by taking the appropriate amount of peptide stock, evaporating the

solvent over a stream of air, addition of HBSS and sonication for 1 min at 55°C.

Liposomes were prepared by using appropriate amounts of lipid and peptide stock

solutions evaporating the solvent over a stream of air, addition of HBSS and

sonication for 1 min at 55°C.

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CHO cell culturing and experiments

Chinese hamster Ovary (CHO) cells were cultivated in Dulbecco’s Modified Eagle’s

Medium (DMEM, PAA-The Cell Culture Company), supplemented with 10% fetal

calf serum (iron supplied), 2% of L-glutamine, 1% penicillin and 1% streptomycin.

The cells were cultured with 5% CO2 at 37 °C. The medium was refreshed every two

days and the cells were passaged by trypsinization at 70% confluence. For

fluorescence assays, the cells were transferred and grown on an 8-well slide (Lab-Tek,

Thermo scientific). One washing step with HBSS was performed. Subsequently, the 5

µM CPK solution was added and left to incubate for 5 minutes at 37 °C. Next, the

liquid was removed and a single washing step was performed. Afterwards, either

fluorescently labeled E peptide (2.5 µM) or 250 µM fluorescently labeled E decorated

liposomes were added to the cells and left to incubate for 5-10 minutes. Subsequently,

the microscope slide was transferred to a Leica SPE confocal microscope and images

were obtained within 1 h. In the CPE incorporation experiment, nuclei were stained

with 100 ng/mL Hoechst 33342 in DMEM for 30 min. 5 µM fluorescently labeled

CPE was added to the wells and cells were imaged over 10 min using an automated

Nikon TiE2000 confocal microscope.

Zebrafish embryo experiments

Zebrafish (Danio rerio) were handled in compliance with the local animal welfare

regulations and maintained according to standard protocols (http://ZFIN.org). After

harvesting embryos from single crosses they were grown at 28°C in egg water

(60μg/ml Instant Ocean sea salts). The stage of embryo was selected based on the auto

fluorescence levels. At one day post fertilization dechorionated embryos were

incubated with the peptides added to the egg water. The experimental group was pre-

treated in 5µM CPK for 30 minutes at 34°C. Next, CPK was replaced with 2.5 µM

complementary fluorescent E peptide and incubated for another 30 minutes at 34°C.

Embryos in the control group were exposed only to 2.5 µM complementary

fluorescent E peptide. Following fluorescent peptides were washed away three times

with egg water; embryos were anesthetized with 200 μg/mL buffered 3-aminobenzoic

acid (Tricaine) and imaged with a Leica M205FA stereo fluorescent microscope.

Docking of liposomes on the biological membranes was also studied using 2 day-old

zebrafish embryos as an in vivo system. 5 µM solution of CPK in egg water was

added to the embryos for 30 minutes incubation at 34°C. After subsequent washing,

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0.25 mM liposomes containing 1 mol% fluorescent DHPE-TR and 1 mol% CPE were

added to the embryos for another 30 minutes at 34°C. Washing and imaging was

performed as mentioned above.

Characterization of fluorescently labeled peptides using HRMS

1. Ac-(EIAALEK)3K-Carboxyfluorescein

Chemical Formula: C131H201N27O41 [2808.45]

HR-MS: Calcd. [1405.225, M+2H]2+, Found 1405.22

2. Ac-(KIAALKE)3K-Carboxyfluorescein


HR-MS: Calcd. [1424.81, M+2H+OAc]2+, Found 1424.817

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Chapter 7

3. Cholesterol-PEG12-(EIAALEK)3K-carboxyfluorescein


HR-MS: Calcd. [1279.05, M+3H]3+, Found 1279.059

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Appendix: supplementary information

Confocal images of CHO cells

A) B) C)

Figure S1. Confocal microscopy images (A: bright field, B: fluorescence and C:

overlay) indicating that peptide E by itself does not bind to the cell membranes. Here,

only fluorescently labeled (carboxyfluorescein) E peptides were added and incubated

for 5 minutes with the CHO cells. Imaging took place after three washes with HBSS.

Experiments performed at 37 °C. The scale bar represents 50 µm.

Figure S2. Confocal microscopy image of CHO cells, showing that the addition of

CPE first, followed by fluorescently labelled K resulted in efficient coiled coil

formation. First, 5 µM CPE (5 µM) was added to the cells for 10 min. Subsequent

addition of fluorescently labeled K peptides (2.5 µM). The image reveals that this

reverse route also yields fluorescence located at the cell membranes, indicating that

coiled coil formation occurred efficiently. All the experiments were performed at 37

°C. The scale bar represents 50 µm.

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Chapter 7

Figure S3. Confocal microscopy images (A: bright field, B: fluorescence and C:

overlay) showing that E decorated liposomes by themselves do not dock onto CHO

cells. CPE liposomes (0.25 mM total lipid concentration) with 1 mol% DHPE-TR

were added to the cells. All the experiments were performed at 37 °C. The scale bar

represents 50 µm.

Stereo microscope images of zebrafish embryos

Figure S4. Control zebrafish embryos exposed only to 2.5 µM complementary

fluorescent E (A: bright field, B: fluorescence, C: overlay). Embryos are shown in

lateral view, anterior to the left.

Figure S5. Control group of zebrafish embryos which was incubated only with 0.25

mM liposomes with 1% CPE and 1% TR for 30 minutes at 34°C (A: bright field, B:

fluorescence, C: overlay). Embryos are shown in lateral view, anterior to the left.

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chapter 7: in vitro and in vivo supramolecular biomembrane

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